Haemophilus influenzae type b has long been recognized as a frequent pathogen, particularly in infants and children, but only recently has nontypable H. influenzae been recognized as an important pathogen. It is now well established that nontypable H. influenzae causes pneumonia, bacteremia, meningitis, postpartum sepsis, and acute febrile tracheobronchitis in adults. In addition, nontypable H. influenzae causes neonatal sepsis and is a frequent etiologic agent in acute otitis media in infants and children. Therefore, the importance of discovering a method to assay a clinical sample such as sputum, cerebral spinal fluid, blood and others for the presence of H. influenzae is clear.
The observation that nontypable H. influenzae causes serious infections in adults and children has stimulated interest in study of the pathogenesis and potential virulence factors associated with this bacterium. The ribitol capsule of H. influenzae type b is a virulence factor for the organism, and antibody to capsule protects the host by means of bactericidal and/or opsonizing actions. These observations have generated much investigation on the role of the capsular polysaccharide in infection with H. influenzae type b and protection from these infections. However, nontypable H. influenzae lacks a polysaccharide capsule, and, similar to the outer membranes of other gram-negative bacteria, the outer membrane of H. influenzae is composed of outer membrane proteins (OMPs) and lipopolysaccharide (LPS). Therefore, studies of the relationship between virulence of nontypable H. influenzae and surface antigens focus on OMPs and LPS.
Analysis of OMPs of nontypable H. influenzae has shown that there are marked differences in OMP composition among strains. See e.g. Murphy et al, "A Subtyping System For Nontypable Haemophilus influenzae Based on Outer-membrane Proteins," J. Infect. Dis, 1983, 147:838-46; Barenkamp et al, "Outer Membrane Protein and Biotype Analysis of Pathogenic Nontypable Haemophilus influenzae," Infect. Immun, 1982, 36:535-40; Loeb et al, "Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae, Pathogenic and Epidemiological Implications," Infect. Immun, 1980, 30:709-17.
A subtyping system for nontypable H. influenzae based on the major OMPs has previously been developed. If a surface exposed antigen (immunogen) which is conserved in all strains could be found, it would be an important tool in developing a method of identifying H. influenzae in clinical specimens as well as a vaccine against H. influenzae. It is therefore an object of this invention to find a surface exposed antigen in both typable and nontypable H. influenzae which is conserved in all strains including typable H. influenzae such as type b which is known to cause bacterial meningitis. It is a further object of this invention to develop a means for predictably identifying such conserved surface exposed antigen. It is a further object to develop a monoclonal antibody against such a surface exposed antigen. A further object of the invention is to develop a means for producing large quantities of such antigen and another object is to isolate and introduce the genetic sequence for such antigen into a novel plasmid and to cause expression of such sequence in a bacteria such as E. coli to produce such antigen.
Another object of the invention is to construct a nucleic acid probe through the combination of the surface exposed antigen in both typable and nontypable H. influenzae which is conserved in all strains and the monoclonal antibody which would be a diagnostic test for detecting H. influenzae.